Thom Jonas, Roters Nathalie, Schuemann Slavica, Andrée Birgit, Buettner Falk F R, Hilfiker Andres, Goecke Tobias, Ramm Robert
Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Hannover Medical School, 30625 Hannover, Germany.
Lower Saxony Centre for Biomedical Engineering, Implant Research and Development, 30625 Hannover, Germany.
Biomedicines. 2024 Jul 3;12(7):1470. doi: 10.3390/biomedicines12071470.
Patterns of humoral immune responses represent a major hurdle in terms of pig-to-human xenotransplantation approaches. The best-known xenogeneic glycan antigens present in pigs are the αGal (Galili antigen) and the non-human sialic acid Neu5Gc. As there are further differences between porcine and human cellular surface glycosylation, a much broader range of glycan epitopes with xeno-reactive relevance can be anticipated. Therefore, we set out to chemically modify porcine cellular surface glycans in a global approach by applying sodium periodate (NaIO) oxidation.
Porcine endothelial cells were exposed to oxidation with 1 to 5 mM NaIO for different time periods at 37 °C or 4 °C and under static or dynamic conditions. The impact on cellular survival was determined by applying live/dead assays. Oxidation of αGal-epitopes was assessed by fluorescence microscopy-based quantification of isolectin-B4 (IL-B4) staining. Overall immunogenicity of porcine cells was determined by human serum antibody binding.
Treatment of porcine endothelial cells and tissues with NaIO led to reduced binding of the αGal-specific IL-B4 and/or human serum antibodies. NaIO was revealed to be cytotoxic when performed at elevated temperatures and for a prolonged time. However, by applying 2 mM NaIO for 60 min at 4 °C, a high extent of cellular viability and a relevant reduction in detectable αGal epitope were observed. No differences were detected irrespectively on whether the cells were oxidized under static or flow conditions.
Glycan epitopes on living cells can be oxidized with NaIO while maintaining their viability. Accordingly, this strategy holds promise to prevent immune reactions mediated by preformed anti-glycan antibodies.
就猪到人的异种移植方法而言，体液免疫反应模式是一个主要障碍。猪体内存在的最著名的异种糖抗原是αGal（加利利抗原）和非人类唾液酸Neu5Gc。由于猪和人类细胞表面糖基化存在进一步差异，可以预期有更广泛的具有异种反应相关性的糖表位。因此，我们着手通过应用高碘酸钠（NaIO）氧化以全局方式化学修饰猪细胞表面聚糖。
将猪内皮细胞在37°C或4°C下、在静态或动态条件下用1至5 mM NaIO氧化不同时间段。通过应用活/死检测法确定对细胞存活的影响。通过基于荧光显微镜的异凝集素-B4（IL-B4）染色定量评估αGal表位的氧化。通过人血清抗体结合确定猪细胞的总体免疫原性。
用NaIO处理猪内皮细胞和组织导致αGal特异性IL-B4和/或人血清抗体的结合减少。当在高温下长时间进行时，NaIO显示具有细胞毒性。然而，通过在4°C下用2 mM NaIO处理60分钟，观察到细胞活力高度维持且可检测到的αGal表位有相应减少。无论细胞是在静态还是流动条件下被氧化，均未检测到差异。
活细胞上的糖表位可用NaIO氧化，同时保持其活力。因此，该策略有望预防由预先形成的抗聚糖抗体介导的免疫反应。
